Adeno-associated viral (AAV) vectors have excellent safety profiles because wild type AAV has never been associated with any human disease. Thus, AAV are popular and successful vectors for gene therapies. AAV vectors have been extensively studied in clinical trials for many different conditions, including haemophilia B, heart disease and congenital blindness. In addition, the first EU licenced gene therapy drug, Glybera, is based on AAV vector for the treatment of familial lipoprotein lipase deficiency (LPLD), exemplifying the potential of AAV vectors in gene therapy.
Although many advances have been made in AAV vector design, barriers such as a pre-existing immune response have necessitated the administration of high titre AAV and, in many cases, a combined administration of an immune-suppressant to achieve clinical efficacy. This presents a significant challenge in AAV production and has considerable safety implications in the clinical use of AAV vectors.
AAV vectors are most commonly produced by a transient co-transfection of AAV plasmids and a helper plasmid derived from another virus, such as an adenovirus. Significant progress has recently been made in large scale production and robust purification of AAV to support clinical development. However, production of high titre AAV is still a significant challenge, requiring patients to receive repeated administrations of a vector to achieve the desired dosage. For example, for Glybera, it is required to administer the vector at a dose of 3×1012 vg/kg via 40 or 60 multiple injections.
Furthermore, as a result of current purification methods, AAV products typically contain high levels of protein aggregates or incompletely packaged empty capsids that lack vector DNA. The empty capsids in final products can often be as high as 40 fold over the level of complete particles. These impurities can trigger unwanted immune responses in patients. For example, recent studies have shown that cellular immune responses in mice and in human are directed to epitopes in the AAV2 capsids, and the presence of empty capsids inhibits hepatocyte transduction in vivo following high dose vector administration. The potential adverse effects of the unwanted immunogenicity of empty capsids compromise product safety and efficacy.
Removal of empty capsids that have no therapeutic function by known methods is difficult due to the innate similarity of their particle size, affinity and protein composition to the complete particles containing vector DNA. There have been continuous efforts to separate empty AAV capsids from genome containing complete particles. Empty particle-free AAV2 has been achieved by differential CHCl3. However, there may be problems using this method in scale up production and GMP manufacture. Ion exchange chromatography has also been reported for the separation of empty capsids in AAV2, 4, 5 and 8. However, from 20% up to 30 fold empty capsids remained in the final products.
Therefore, there is need to develop new methods of AAV vector production which decrease or eliminate the presence of empty capsids in the final product. This would improve the safety and efficacy of AAV products. Reduction of empty particles would also overcome the hurdle in high titre production.